Zace1: a human metalloenzyme

ABSTRACT

Angiotensin-converting enzyme is a zinc metallopeptidase that plays roles in blood pressure regulation and fertility. The catalytic activities of angiotensin converting enzymes include the production of the potent vasopressor angiotensin II from angiotensin I, and the inactivation of the vasodilatory peptide bradykinin. Zace1 is a new form of human zinc metallopeptidase, which includes one zinc-dependent catalytic domain containing the motif “HEXXH” and one downstream “EX(I/V)X(D/S)” motif.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 09/846,996, filed May 1, 2001, now U.S. Pat. No. 6,524,836, which isa divisional of U.S. patent application Ser. No. 09/440,325, filed Nov.15, 1999, now U.S. Pat. No. 6,280,994, which claims the benefit of U.S.Patent Application Ser. No. 60/109,783, filed Nov. 25, 1998 all of whichare herein incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to a new protein expressed byhuman cells. In particular, the present invention relates to a novelgene that encodes a metalloenzyme, designated as “Zace1,” and to nucleicacid molecules encoding Zace1 polypeptides.

BACKGROUND OF THE INVENTION

Angiotensin-converting enzyme (ACE; peptidyl dipeptidase A; kininase II(EC 3.4.15.1)) is a zinc metallopeptidase that plays roles in bloodpressure regulation and fertility. ACE is rather nonspecific and cleavesdipeptides from a broad range of substrates. In general, ACE cleaves aC-terminal dipeptide “A-B” from a polypeptide when A is not a prolineresidue, and B is neither an aspartate nor a glutamate residue. Forexample, ACE cleaves a single C-terminal dipeptide from angiotensin I toproduce the potent vasopressor angiotensin II, and ACE cleaves theC-terminal dipeptide from [des-Asp¹]angiotensin I to produce angiotensinIII. The enzyme also inactivates the vasodilatory peptide bradykinin bysequential removal of two C-terminal dipeptides. For a general review ofangiotensin-converting enzyme, see Corvol et al., Meth. Enzymol. 246:283(1995), Corvol et al., J. Hypertension 13(Suppl. 3):S3 (1995), Jacksonand Garrison, “Renin and Angiotensin,” in Goodman and Gilman's ThePharmaceutical Basis of Therapeutics, 9^(th) Edition, Molinoff andRuddon (eds.), pages 733-758 (McGraw-Hill 1996), Matsusaka and Ichikawa,Annu. Rev. Physiol. 59:395 (1997), and Zimmerman and Dunham, Annu. Rev.Pharmacol. Toxicol. 37:53 (1997).

ACE is a cleavable ectoprotein anchored to the plasma membrane through atransmembrane domain. The majority of the membrane-bound form isextracellularly exposed, and this extracellular domain includes at leastone active site. A soluble form of ACE circulates in plasma (see, forexample, Hooper and Turner, Biochem. Soc. Trans., 17:660 (1989)).

Two ACE isoforms have been identified in mammalian tissues. Thepredominant form is referred to as “somatic” ACE, which has a molecularweight of about 150 kD to about 180 kD, and is predominantly found atthe surface of vascular endothelial cells, epithelial cells, andneuroepithelial cells. The other isoform is referred to as “germinal”ACE or testis ACE (tACE), which has a molecular weight of about 90 kD toabout 110 kD, and is expressed in post-meiotic cells and sperm. Humansomatic ACE has two homologous domains, each comprising a catalytic siteand a Zn⁺²-binding region, while human testis ACE contains one catalyticcite.

Hubert et al., J. Biol. Chem. 266:15377 (1991), describe the completeintron-exon structure of the human ACE gene. The human ACE gene contains26 exons, wherein exon 1 to exon 26 is transcribed in somatic ACE mRNA,but exon 13 is removed by splicing; germinal ACE mRNA is transcribedfrom exon 13 to exon 26. Exons 4-11 and 17-24 encode the two homologousdomains (N domain and C domain) within somatic ACE, and are very similarin size and structure. The intron sizes are not conserved. Since somaticACE and tACE are transcribed from a single gene, alternate splicing oralternative start sites for transcription initiation may be involved.Two functional promoters reside within the gene, which would supportinitiation from distinct start sites under separate control. The tACEpromoter is upstream of the 5′ end of tACE mRNA, with a transcriptionalinitiation

Inhibitors of angiotensin-converting enzyme are used for the treatmentof hypertension of various conditions, including left ventricularsystolic dysfunction, progressive renal impairment, scleroderma renalcrisis, congestive heart failure due to systolic dysfunction, andtreatment of atherosclerosis (see, for example, Brown and Vaughan,Circulation 97:1411 (1998); Mancini, Am. J. Med. 105:40S (1998);Parmley, Am. J. Med. 105:27S (1998)). There are at least nine ACEinhibitors approved for use in the United States.

ACE inhibitors can be classified into at least three groups: (1)sulfhydryl-containing inhibitors structurally related to captopril(e.g., fentiapril, pivalopril, zofenopril, alacepril), (2)dicarboxyl-containing inhibitors structurally related to enalapril(e.g., lisinopril, benazepril, quinapril, moexipril, ramipril,spirapril, perindopril, indolapril, pentopril, indalapril, cilazapril),and (3) phosphorus-containing inhibitors structurally related tofosinopril. New classes of ACE inhibitors are sought that will inhibitACE and other zinc metalloproteases. Moreover, new types of ACEinhibitors are also sought that will selectively inhibit ACE hydrolysisof N-acetyl-seryl-aspartyl-lysyl-prolyl (AcSDKP), a regulatory factor inhematopoiesis, without effect on angiotensin I or bradykinin metabolism.

Thus, a continuing need exists for the characterization of new forms ofzinc metallopeptidases, and the use of the enzymes to identifytherapeutically useful compounds.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel metallopeptidase, designated“Zace1.” The present invention also provides Zace1 polypeptides andZace1 fusion proteins, nucleic acid molecules encoding such polypeptidesand proteins, and methods for using these nucleic acid molecules andamino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

A Zace1 polypeptide has the following amino acid sequence: MGARWTCCPGPSLLVLLCYG QLLPWLRIKG EHSLGVAGTP RSMGPDKGTG CNETETKIFL QFYDQTGEVVLNKFMEATWN YVTNITRKNQ EEMMKDMERS QFMIYFGTQA HLFKVTQFKD PDVNGMLSKLQNIDKAALSK DELREYNELL AXLEMTYSMA QVCLNEGPCL SLESELEVMA TSRDKEELLWAWQGWQDAVG RQICTTFEHY VELSNKAAQL NGVXKDMGAL WHSKYESDTL EQDLERLFQELRPLYLNPHT YVRRALHRHY GPELIDLRGP IPAHLLGENT LAQSWVNILD PVLPFLKKIPEDVTKIMKVQ HWKPEKLMLE EAETFFTYLG LALPPAPPSF WKKLMLMRPT DGREVECHISAWNFYQDDDF RIKKCAEVTT EDPLSIFHEM GHFQYFLQYK NLSIIFRTGA NPAFEEAVGSVITLSASSHK HLLNIGLLSL LEDEVNFLMH IALEKIAFIP FGYLMDLFRW KVFDGTIWKDIYNQEWWNLR RLKYQGLCPA IPHSEEDFDP GAKFHFSAGV PYIRRYFLSL VLQFQFHETLCKASGHMGPL HQCDIYNSKI AGKLLALKLG SSKPWPEVLK MLTGESEVST NVFMTYFKPLLTWLVTEHAA RGETPGVPLQ FYPPYETPMS STEKDTDKVT FLSLKLDPNQ AKFGYWVLLALGFVMSLVVL GLACRLQSLE KQSL (SEQ ID NO:1).

Zace1 is a new zinc metalloprotease that is a paralog of testis ACE.Somatic ACE and testis ACE (also called germinal ACE) are ACE isoformsthat exhibit similar enzymatic activities. Human endothelial somatic ACEhas 1306 amino acid residues, including 14 cysteine residues and 17potential N-linked glycosylation sites, but no Ser/Thr-rich regionindicative of O-linked glycosylation sites. Additional features of ACEinclude a 29 amino acid residue hydrophobic signal peptide, and a 17amino acid residue transmembrane domain. Somatic ACE also contains twohomologous domains, designated as the “N domain” and the “C domain.” Thecurrent belief is that these duplicative domains arose from duplicationof an ancestral gene. The overall amino acid sequence similarity betweenthe N domain and C domain of somatic ACE is about 60%, but is about 89%with respect to 40 amino acids in each domain that include the residuesof each active site. The number and relative position of the Cysresidues in the two domains is identical. There is little or no sequencesimilarity between the amino-terminal and carboxy-terminal portions ofsomatic ACE, and little or no sequence similarity between the stretch ofresidues that connects the N and C domains of somatic ACE. Each of the Nand C domains contains a zinc-binding motif His-Glu-Xaa-Xaa-His (HEXXH)that is present in many zinc metalloproteases. The two His residues ofthis motif within somatic ACE provide two of the three zinc-coordinatingligands; the third zinc-coordinating ligand is a Glu residue downstreamof the C domain's HEXXH C-terminal His.

Zinc is essential for the catalytic activity of ACE, and the zinc ion ispredicted to function directly in the catalytic step of peptidehydrolysis by polarizing the zinc-bound water molecule, which theninitiates nucleophilic attack on the substrate carbonyl scissile bond.Thus, ACE is a member of the thermolysin branch of the zincmetalloproteases. Monovalent anions enhance the enzymatic hydrolysis ofsome, but not all, ACE substrates. For some substrates, a concommitantincrease in pH increases the amount of monovalent anion (such aschloride) stimulation.

Germinal ACE (testis ACE; tACE) has 732 amino acid residues, andcorresponds to the C domain of somatic ACE. Thus, tACE has only oneactive site, only one HEXXH motif, and a third zinc-coordinating ligandthat is a Glu residue 23 residues downstream of the C-terminal histidineresidue of the HEXXH motif. The N-terminal 67 amino acid residues oftACE are specific to tACE, and contain a signal peptide that is distinctfrom that of somatic ACE, as well as a Ser/Thr-rich region forO-glycosylation. Somatic ACE and tACE mRNAs are transcribed from asingle gene.

Zace1 contains 694 amino acid residues, whereas mammalian tACE contains732 amino acid residues. When polypeptides Zace1 and tACE are aligned(with gap residues inserted in Zace1 and tACE to provide appropriatealignment), Zace1 exhibits 53% amino acid sequence identity to tACE. Theseven, highly conserved Cys residues present in tACE and the C domain ofsomatic ACE are present in Zace1 (at residues 163, 169, 367, 385, 508,551 and 563). The loops that would be formed by the predicted disulfidebonding pairs of Zace1 (residues 163 to 169, residues 367 to 385, andresidues 551 to 563) correspond to the three loops of 5, 17 and 11residues found in tACE. In addition, Zace1 has two additional Cysresidues, at positions 51 and 204. The zinc-binding motif HEXXH ispresent at residues 398 to 402 of Zace1. An expanded zinc binding regionsignature of zinc metallopeptidases has the following sequence:[GSTALIVN]-x-x-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ], where “x” isany amino acid residue, acceptable amino acid residues are listedbetween square brackets, and unacceptable amino acid residues are listedbetween braces (PROSITE sequence No. PS00142 of Release 15.0; Bairoch etal., Nucleic Acids Res. 24:217 (1997)). This signature resides withinthe Zace1 polypeptide at amino acid residues 395 to 404 of SEQ ID NO:1.

In Zace1, 23 residues separate the C-terminal His of the HEXXH motif andthe conserved EX(I/V)X(D/S) motif present at residues 426 to 430, where“(I/V)” indicates that either I or V may be present and “(D/S)”indicates that either D or S may be present. The Glu domain at position426 is predicted to be the third zinc-binding (or zinc-coordinating)ligand within Zace1. At position 430, Zace1 has a serine residuesubstituted for an aspartic acid residue, which occurs at acorresponding position in ACE. The transmembrane domain of Zace1includes amino acid residues 663 to 684 of SEQ ID NO:1

As described below, the present invention provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90% identical to a reference amino acid sequence selected from thegroup consisting of: (a) amino acid residues 367 to 430 of SEQ ID NO:1,(b) amino acid residues 163 to 563 of SEQ ID NO:1, (c) amino acidresidues 52 to 563 of SEQ ID NO:1, (d) amino acid residues 52 to 644 ofSEQ ID NO:1, (e) amino acid residues 52 to 648 of SEQ ID NO:1, (f) aminoacid residues 52 to 655 of SEQ ID NO:1, (g) amino acid residues 52 to662 of SEQ ID NO:1, (h) amino acid residues 52 to 682 of SEQ ID NO:1,(i) amino acid residues 52 to 694 of SEQ ID NO:1, and (j) amino acidresidues 1 to 694 of SEQ ID NO:1, wherein the isolated polypeptideeither (i) specifically binds with an antibody that specifically bindswith a polypeptide consisting of the amino acid sequence of SEQ ID NO:1,or (ii) exhibits dipeptidyl carboxypeptidase activity.

Illustrative polypeptides include polypeptides comprising an amino acidsequence selected from the group consisting of: (a) amino acid residues367 to 430 of SEQ ID NO:1, (b) amino acid residues 163 to 563 of SEQ IDNO:1, (c) amino acid residues 52 to 563 of SEQ ID NO:1, (d) amino acidresidues 52 to 644 of SEQ ID NO:1, (e) amino acid residues 52 to 648 ofSEQ ID NO:1, (f) amino acid residues 52 to 655 of SEQ ID NO:1, (g) aminoacid residues 52 to 662 of SEQ ID NO:1, (h) amino acid residues 52 to682 of SEQ ID NO:1, (i) amino acid residues 52 to 694 of SEQ ID NO:1,and (j) amino acid residues 1 to 694 of SEQ ID NO:1. Such a polypeptidecan be a metallopeptidase.

Additional exemplary polypeptides include polypeptides that comprise anamino acid sequence comprising the motif“[GSTALIVN]-x-x-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ],” where “x” isany amino acid residue, acceptable amino acid residues are listedbetween square brackets, and unacceptable amino acid residues are listedbetween braces. For example, an illustrative polypeptide comprises aminoacid residues 395 to 404 of SEQ ID NO:1.

The present invention also provides isolated polypeptides comprising anextracellular domain, wherein the extracellular domain comprises aminoacid residues 52 to 662 of the amino acid sequence of SEQ ID NO:1. Suchpolypeptides can further comprise a transmembrane domain that resides ina carboxyl-terminal position relative to the extracellular domain,wherein the transmembrane domain comprises amino acid residues 663 to684 of SEQ ID NO:1. These polypeptides can also comprise anintracellular domain that resides in a carboxyl-terminal positionrelative to the transmembrane domain, wherein the intracellular domaincomprises amino acid residues 685 to 694 of SEQ ID NO:1. Suchpolypeptides can also include a signal secretory sequence that residesin an amino-terminal position relative to the extracellular domain. Asignal secretory sequence is provided by amino acid residues 1 to 51 ofthe amino acid sequence of SEQ ID NO:1.

The present invention also includes variant Zace1 polypeptides, whereinthe amino acid sequence of the variant polypeptide shares an identitywith the amino acid sequence of SEQ ID NO:1 selected from the groupconsisting of at least 70% identity, at least 80% identity, at least 90%identity, at least 95% identity, or greater than 95% identity, andwherein any difference between the amino acid sequence of the variantpolypeptide and the amino acid sequence of SEQ ID NO:1 is due to one ormore conservative amino acid substitutions. In addition, the presentinvention contemplates isolated polypeptides, consisting of an aminoacid sequence selected from the group consisting of: (a) amino acidresidues 367 to 430 of SEQ ID NO:1, (b) amino acid residues 163 to 563of SEQ ID NO:1, (c) amino acid residues 52 to 563 of SEQ ID NO:1, (d)amino acid residues 52 to 644 of SEQ ID NO:1, (e) amino acid residues 52to 648 of SEQ ID NO:1, (f) amino acid residues 52 to 655 of SEQ ID NO:1,(g) amino acid residues 52 to 662 of SEQ ID NO:1, (h) amino acidresidues 52 to 682 of SEQ ID NO:1, (i) amino acid residues 52 to 694 ofSEQ ID NO:1, and (j) amino acid residues 1 to 694 of SEQ ID NO:1.

The present invention also contemplate allelic variants and orthologs ofthe Zace1 polypeptides described herein.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units.

The present invention also provides isolated nucleic acid molecules thatencode a polypeptide comprising an amino acid sequence selected from thegroup consisting of: (a) amino acid residues 367 to 430 of SEQ ID NO:1,(b) amino acid residues 163 to 563 of SEQ ID NO:1, (c) amino acidresidues 52 to 563 of SEQ ID NO:1, (d) amino acid residues 52 to 644 ofSEQ ID NO:1, (e) amino acid residues 52 to 648 of SEQ ID NO:1, (f) aminoacid residues 52 to 655 of SEQ ID NO:1, (g) amino acid residues 52 to662 of SEQ ID NO:1, (h) amino acid residues 52 to 682 of SEQ ID NO:1,(i) amino acid residues 52 to 694 of SEQ ID NO:1, and (j) amino acidresidues 1 to 694 of SEQ ID NO:1. An illustrative nucleic acid moleculeencodes amino acid residues 1 to 694 of SEQ ID NO:1.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors cancomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells and recombinant viruses comprising these vectorsand expression vectors. Illustrative host cells include bacterial,yeast, fungal, insect, mammalian, and plant cells. Recombinant hostcells comprising such expression vectors can be used to produce Zace1polypeptides by culturing such recombinant host cells that comprise theexpression vector and that produce the Zace1 protein, and, optionally,isolating the Zace1 protein from the cultured recombinant host cells.

The present invention further includes fusion proteins comprising aZace1 polypeptide or peptide, and nucleic acid molecules that encodesuch fusion proteins.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a Zace1 polypeptide or Zace1 antibody described herein.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule. For example, representativecontigs to the polynucleotide sequence 5′ ATGGAGCTT 3′ are 5′ AGCTTgagt3′ and 3′ tcgacTACC 5′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

The term “isolated genomic DNA” denotes DNA obtained from the genome ofa cell that contains exons, introns and nontranscribed DNA.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces Zace1from an expression vector. In contrast, Zace1 can be produced by a cellthat is a “natural source” of Zace1, and that lacks an expressionvector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zace1polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of Zace1using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-Zace1antibody, and thus, an anti-idiotype antibody mimics an epitope ofZace1.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zace1 monoclonal antibody fragment bindswith an epitope of Zace1.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a Zace1polypeptide component. Examples of an antibody fusion protein is aprotein that comprises a Zace1 catalytic domain and either an Fc domainor an antigen-biding region.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zace1” or a “Zace1anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zace1 gene, or(b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zace1 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant Zace1 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:1. Such variants include naturally-occurringpolymorphisms of Zace1 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:1.

Alternatively, variant Zace1 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zace1gene or variant Zace1 polypeptide, a variant gene or polypeptide encodedby a variant gene may be functionally characterized the ability to bindspecifically to an anti-Zace1 antibody or by the dipeptidase (e.g.,dipeptidyl carboxypeptidase) activity of the variant Zace1 polypeptide.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zace1 genes.Within the context of this invention, a “functional fragment” of a Zace1gene refers to a nucleic acid molecule that encodes a portion of a Zace1polypeptide which either has peptidyl dipeptidase activity orspecifically binds with an anti-Zace1 antibody.

The term “dipeptidyl peptidase” refers to an enzyme that cleavesdipeptides from the amino terminus of a polypeptide, whereas the term“dipeptidyl carboxypeptidase” refers to an enzyme that cleavesdipeptides from the carboxyl terminus of a polypeptide.

A “metallopeptidase” is a peptide hydrolase which uses a metal in thecatalytic mechanism. Typically, metallopeptidases contain a tightlybound transition metal, such as zinc or iron. Angiotensin-convertingenzyme (ACE) is an example of a zinc metallopeptidase. The enzymaticactivities of ACE include cleavage of the carboxyl-terminal dipeptidefrom angiotensin I to produce angiotensin II, removal of twocarboxyl-terminal dipeptides from bradykinin, hydrolysis ofN-acetyl-Ser-Gly-Lys-Pro at the Gly-Lys bond, cleavage of acarboxyl-terminal tripeptide amide from substance P, and luteinizinghormone releasing hormone, and an amino-terminal tripeptide fromluteinizing hormone releasing hormone. Several examples of artificialACE substrate are described herein.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of Zace1 Polynucleotides

Nucleic acid molecules encoding a human Zace1 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon the amino acid sequence of SEQ ID NO:1. These techniques arestandard and well-established.

As an illustration, a nucleic acid molecule that encodes a human Zace1gene can be isolated from a cDNA library. In this case, the first stepwould be to prepare the cDNA library by isolating RNA from a tissue,using methods well-known to those of skill in the art. In general, RNAisolation techniques must provide a method for breaking cells, a meansof inhibiting RNase-directed degradation of RNA, and a method ofseparating RNA from DNA, protein, and polysaccharide contaminants. Forexample, total RNA can be isolated by freezing tissue in liquidnitrogen, grinding the frozen tissue with a mortar and pestle to lysethe cells, extracting the ground tissue with a solution ofphenol/chloroform to remove proteins, and separating RNA from theremaining impurities by selective precipitation with lithium chloride(see, for example, Ausubel et al. (eds.), Short Protocols in MolecularBiology, 3^(rd) Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)[“Ausubel (1995)”]; Wu et al., Methods in Gene Biotechnology, pages33-41 (CRC Press, Inc. 1997) [“Wu (1997)”]). Alternatively, total RNAcan be isolated by extracting ground tissue with guanidiniumisothiocyanate, extracting with organic solvents, and separating RNAfrom contaminants using differential centrifugation (see, for example,Chirgwin et al., Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 cells, which can beobtained, for example, from Life Technologies, Inc. (Gaithersburg, Md.).

A human genomic library can be prepared by means well-known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes, which have a nucleotide sequence based uponthe amino acid sequence of SEQ ID NO:1, using standard methods (see, forexample, Ausubel (1995) at pages 6-1 to 6-11).

Anti-Zace1 antibodies, produced as described below, can also be used toisolate DNA sequences that encode human Zace1 genes from cDNA libraries.For example, the antibodies can be used to screen λgt11 expressionlibraries, or the antibodies can be used for immunoscreening followinghybrid selection and translation (see, for example, Ausubel (1995) atpages 6-12 to 6-16; Margolis et al., “Screening λ expression librarieswith antibody and protein probes,” in DNA Cloning 2: Expression Systems,2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press1995)).

The sequence of a Zace1 cDNA or Zace1 genomic fragment can be determinedusing standard methods. Promoter elements from a Zace1 gene can be usedto direct the expression of heterologous genes in transgenic animals orpatients treated with gene therapy. The identification of genomicfragments containing a Zace1 promoter or regulatory element can beachieved using well-established techniques, such as deletion analysis(see, generally, Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zace1proteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zace1 gene in a cell is altered byintroducing into the Zace1 locus a DNA construct comprising at least atargeting sequence, a regulatory sequence, an exon, and an unpairedsplice donor site. The targeting sequence is a Zace1 5′ non-codingsequence that permits homologous recombination of the construct with theendogenous Zace1 locus, whereby the sequences within the constructbecome operably linked with the endogenous Zace1 coding sequence. Inthis way, an endogenous Zace1 promoter can be replaced or supplementedwith other regulatory sequences to provide enhanced, tissue-specific, orotherwise regulated expression.

4. Production of Zace1 Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, that encode the Zace1 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:2is a degenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the Zace1 polypeptide of SEQ ID NO:1. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:2 also provides all RNA sequences encoding SEQ ID NO:1, bysubstituting U for T.

Table 1 sets forth the one-letter codes used within SEQ ID NO:2 todenote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D  A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:2, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:1. Variant sequences can be readily tested forfunctionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequence disclosed in SEQ ID NO:2 serves as a templatefor optimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zace1 polypeptides fromother mammalian species, including mouse, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. Orthologs ofhuman Zace1 can be cloned using information and compositions provided bythe present invention in combination with conventional cloningtechniques. For example, a Zace1 cDNA can be cloned using mRNA obtainedfrom a tissue or cell type that expresses the Zace1 polypeptidedisclosed herein. A library is then prepared from mRNA of a positivetissue or cell line.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human Zace1, and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the nucleotide sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:1. cDNA molecules generated from alternatively spliced mRNAs, whichretain the properties of the Zace1 polypeptide are included within thescope of the present invention, as are polypeptides encoded by suchcDNAs and mRNAs. Allelic variants and splice variants of these sequencescan be cloned by probing cDNA or genomic libraries from differentindividuals or tissues according to standard procedures known in theart.

Isolated polynucleotides that encode the Zace1 polypeptide of SEQ IDNO:1 will hybridize to similar sized regions of a related (homologous)polynucleotide, or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is up toabout 0.03 M at pH 7 and the temperature is at least about 60° C.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). Typically, hybridization buffers containfrom between 10 mM-1 M Na⁺. The addition of destabilizing or denaturingagents such as formamide, tetralkylammonium salts, guanidinium cationsor thiocyanate cations to the hybridization solution will alter theT_(m) of a hybrid. Typically, formamide is used at a concentration of upto 50% to allow incubations to be carried out at more convenient andlower temperatures. Formamide also acts to reduce non-specificbackground when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant Zace1polypeptide can be hybridized with a nucleic acid molecule comprising anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:1at 42° C. overnight in a solution comprising 50% formamide, 5×SSC, 50 mMsodium phosphate (pH 7.6), 5× Denhardt's solution (100× Denhardt'ssolution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2%(w/v) bovine serum albumin), 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA. One of skill in the art can devisevariations of these hybridization conditions. For example, thehybridization mixture can be incubated at a higher temperature, such asabout 65° C., in a solution that does not contain formamide. Moreover,premixed hybridization solutions are available (e.g., EXPRESSHYBHybridization Solution from CLONTECH Laboratories, Inc.), andhybridization can be performed according to the manufacturer'sinstructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant Zace1 polypeptide hybridize with a nucleic acidmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO:1 under stringent washing conditions, in which thewash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.including 0.5×SSC with 0.1% SDS at 55° C. or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantZace1 polypeptide hybridize with a nucleic acid molecule comprising anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:1under highly stringent washing conditions, in which the wash stringencyis equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated Zace1 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:1, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the sequences shown in SEQ ID NO:1, or theirorthologs.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZace1 variant. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:1) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom four to six.

The present invention includes polypeptides having one or moreconservative amino acid changes, compared with the amino acid sequenceof SEQ ID NO:1. That is, variants can be obtained that contain one ormore amino acid substitutions of SEQ ID NO:1, in which an alkyl aminoacid is substituted for an alkyl amino acid in a Zace1 amino acidsequence, an aromatic amino acid is substituted for an aromatic aminoacid in a Zace1 amino acid sequence, a sulfur-containing amino acid issubstituted for a sulfur-containing amino acid in a Zace1 amino acidsequence, a hydroxy-containing amino acid is substituted for ahydroxy-containing amino acid in a Zace1 amino acid sequence, an acidicamino acid is substituted for an acidic amino acid in a Zace1 amino acidsequence, a basic amino acid is substituted for a basic amino acid in aZace1 amino acid sequence, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid in a Zace1 aminoacid sequence. Among the common amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Particular variants of Zace1 are characterized by having at least 70%,at least 80%, at least 90%, at least 95% or greater than 95% sequenceidentity to the corresponding amino acid sequence (i.e., SEQ ID NO:1),wherein the variation in amino acid sequence is due to one or moreconservative amino acid substitutions.

Conservative amino acid changes in a Zace1 gene can be introduced, forexample, by nucleotide substitution into a nucleotide sequence thatencodes the amino acid sequence SEQ ID NO:1. Such “conservative aminoacid” variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like (see Ausubel (1995) at pages8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)). A variant Zace1 polypeptide can beidentified by the ability to specifically bind anti-Zace1 antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zace1 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

As discussed above, amino acid sequence analysis indicates that thefollowing amino acids play a role in Zace1 enzymatic activity: His³⁹⁸,His⁴⁰², and Glu⁴²⁶. Although sequence analysis can be used to furtherdefine the Zace1 active site, domains that play a role in Zace1 activitycan also be determined by physical analysis of structure, as determinedby such techniques as nuclear magnetic resonance, crystallography,electron diffraction or photoaffinity labeling, in conjunction withmutation of putative contact site amino acids. See, for example, de Voset al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed Zace1 nucleotide and polypeptide sequences canalso be generated through DNA shuffling as disclosed by Stemmer, Nature370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747 (1994), andinternational publication No. WO 97/20078. Briefly, variant DNAmolecules are generated by in vitro homologous recombination by randomfragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zace1 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zace1polypeptides and nucleic acid molecules encoding such functionalfragments. Functional analysis of the amino acid sequence describedherein can be performed using standard techniques. For example, studieson the truncation at either or both termini of interferons have beensummarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995).Moreover, standard techniques for functional analysis of proteins aredescribed by, for example, Treuter et al., Molec. Gen. Genet. 240:113(1993), Content et al., “Expression and preliminary deletion analysis ofthe 42 kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Illustrative functional fragments include polypeptides, comprising aminoacid residues 367 to 430 of SEQ ID NO:1, amino acid residues 163 to 563of SEQ ID NO:1, amino acid residues 52 to 563 of SEQ ID NO:1, amino acidresidues 52 to 644 of SEQ ID NO:1, amino acid residues 52 to 648 of SEQID NO:1, amino acid residues 52 to 655 of SEQ ID NO:1, amino acidresidues 52 to 662 of SEQ ID NO:1, amino acid residues 52 to 682 of SEQID NO:1, or amino acid residues 52 to 694 of SEQ ID NO:1, and the like.Particular functional fragments of a Zace1 polypeptide are soluble formsof Zace1 that lack a transmembrane domain. Illustrative Zace1 solubleforms include polypeptides consisting of amino acid residues 1 to 662 ofSEQ ID NO:1, amino acid residues 52 to 662 of SEQ ID NO:1, and the like.

The present invention also contemplates functional fragments of a Zace1gene that have amino acid changes, compared with the amino acid sequenceof SEQ ID NO:1. A variant Zace1 gene can be identified on the basis ofstructure by determining the level of identity with the amino acidsequence of SEQ ID NO:1, as discussed above.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zace1 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fifteen amino acids,or about 15 to about 30 amino acids of SEQ ID NO:1. Such epitope-bearingpeptides and polypeptides can be produced by fragmenting a Zace1polypeptide, or by chemical peptide synthesis, as described herein.Moreover, epitopes can be selected by phage display of random peptidelibraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)).Standard methods for identifying epitopes and producing antibodies fromsmall peptides that comprise an epitope are described, for example, byMole, “Epitope Mapping,” in Methods in Molecular Biology, Vol. 10,Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,“Production and Characterization of Synthetic Peptide-DerivedAntibodies,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 60-84 (CambridgeUniversity Press 1995), and Coligan et al. (eds.), Current Protocols inImmunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons1997).

For any Zace1 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zace1 variants based upon the nucleotideand amino acid sequences described herein. Accordingly, the presentinvention includes a computer-readable medium encoded with a datastructure that provides at least one of the following sequences: SEQ IDNO:1, and SEQ ID NO:2. Suitable forms of computer-readable media includemagnetic media and optically-readable media. Examples of magnetic mediainclude a hard or fixed drive, a random access memory (RAM) chip, afloppy disk, digital linear tape (DLT), a disk cache, and a ZIP disk.Optically readable media are exemplified by compact discs (e.g., CD-readonly memory (ROM), CD-rewritable (RW), and CD-recordable), and digitalversatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

5. Production of Zace1 Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zace1 gene, a nucleic acid molecule encoding the polypeptide must beoperably linked to regulatory sequences that control transcriptionalexpression in an expression vector and then, introduced into a hostcell. In addition to transcriptional regulatory sequences, such aspromoters and enhancers, expression vectors can include translationalregulatory sequences and a marker gene which is suitable for selectionof cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zace1 expression vector maycomprise a Zace1 gene and a secretory sequence derived from any secretedgene.

Zace1 proteins of the present invention may be expressed in mammaliancells. Examples of suitable mammalian host cells include African greenmonkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin etal., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zace1 gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. Preferably, the transfected cells areselected and propagated to provide recombinant host cells that comprisethe expression vector stably integrated in the host cell genome.Techniques for introducing vectors into eukaryotic cells and techniquesfor selecting such stable transformants using a dominant selectablemarker are described, for example, by Ausubel (1995) and by Murray(ed.), Gene Transfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A preferred amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zace1 polypeptides can also be produced by cultured mammalian cellsusing a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, vaccinia virus and adeno-associatedvirus (AAV). Adenovirus, a double-stranded DNA virus, is currently thebest studied gene transfer vector for delivery of heterologous nucleicacid (for a review, see Becker et al., Meth. Cell Biol. 43:161 (1994),and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages ofthe adenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

Zace1 can also be expressed in other higher eukaryotic cells, such asavian, fungal, insect, yeast, or plant cells. The baculovirus systemprovides an efficient means to introduce cloned Zace1 genes into insectcells. Suitable expression vectors are based upon the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV), and containwell-known promoters such as Drosophila heat shock protein (hsp) 70promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zace1 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zace1 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zace1 gene is transformed into E. coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native Zace1 secretory signal sequences with secretorysignal sequences derived from insect proteins. For example, a secretorysignal sequence from Ecdysteroid Glucosyltransferase (EGT), honey beeMelittin (Invitrogen Corporation; Carlsbad, Calif.), or baculovirus gp67(PharMingen: San Diego, Calif.) can be used in constructs to replace thenative Zace1 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zace1 genes can be expressed in prokaryotic host cells.Suitable promoters that can be used to express Zace1 polypeptides in aprokaryotic host are well-known to those of skill in the art and includepromoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, theP_(R) and P_(L) promoters of bacteriophage lambda, the trp, recA, heatshock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ promoters of E. coli,promoters of B. subtilis, the promoters of the bacteriophages ofBacillus, Streptomyces promoters, the int promoter of bacteriophagelambda, the bla promoter of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene. Prokaryotic promoters have beenreviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et al.,Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and byAusubel et al. (1995).

Preferred prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a Zace1 polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al, Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287:34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, preferably at least nine, and more preferably at least 15contiguous amino acid residues of SEQ ID NO:1. Within certainembodiments of the invention, the polypeptides comprise 20, 30, 40, 50,100, or more contiguous residues of SEQ ID NO:1. Nucleic acid moleculesencoding such peptides and polypeptides are useful as polymerase chainreaction primers and probes.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

6. Production of Zace1 Fusion Proteins and Conjugates

Fusion proteins of Zace1 can be used to express Zace1 in a recombinanthost, and to isolate the produced Zace1. As described below, particularZace1 fusion proteins also have uses in diagnosis and therapy.

One type of fusion protein comprises a peptide that guides a Zace1polypeptide from a recombinant host cell. To direct a Zace1 polypeptideinto the secretory pathway of a eukaryotic host cell, a secretory signalsequence (also known as a signal peptide, a leader sequence, preprosequence or pre sequence) is provided in the Zace1 expression vector.While the secretory signal sequence may be derived from Zace1, asuitable signal sequence may also be derived from another secretedprotein or synthesized de novo. The secretory signal sequence isoperably linked to a Zace1-encoding sequence such that the two sequencesare joined in the correct reading frame and positioned to direct thenewly synthesized polypeptide into the secretory pathway of the hostcell. Secretory signal sequences are commonly positioned 5′ to thenucleotide sequence encoding the polypeptide of interest, althoughcertain secretory signal sequences may be positioned elsewhere in thenucleotide sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Although the secretory signal sequence of Zace1 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of Zace1 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone α-factor (encoded by the MFα1 gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,for example, Romanos et al., “Expression of Cloned Genes in Yeast,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zace1 canbe expressed as a fusion protein comprising a glutathione S-transferasepolypeptide. Glutathione S-transferease fusion proteins are typicallysoluble, and easily purifiable from E. coli lysates on immobilizedglutathione columns. In similar approaches, a Zace1 fusion proteincomprising a maltose binding protein polypeptide can be isolated with anamylose resin column, while a fusion protein comprising the C-terminalend of a truncated Protein A gene can be purified using IgG-Sepharose.Established techniques for expressing a heterologous polypeptide as afusion protein in a bacterial cell are described, for example, byWilliams et al., “Expression of Foreign Proteins in E. coli UsingPlasmid Vectors and Purification of Specific Polyclonal Antibodies,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(Eds.), pages 15-58 (Oxford University Press 1995). In addition,commercially available expression systems are available. For example,the PINPOINT Xa protein purification system (Promega Corporation;Madison, Wis.) provides a method for isolating a fusion proteincomprising a polypeptide that becomes biotinylated during expressionwith a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

Another form of fusion protein comprises a Zace1 polypeptide and animmunoglobulin heavy chain constant region, typically an F_(c) fragment,which contains two or three constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et al., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:3). In this fusion protein, a preferred Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zace1 fusion protein that comprises a Zace1 moiety and ahuman Fc fragment, wherein the C-terminus of the Zace1 moiety isattached to the N-terminus of the Fc fragment via a peptide linker, suchas a peptide consisting of the amino acid sequence of SEQ ID NO:3. TheZace1 moiety can be a Zace1 molecule or a fragment thereof. For example,a fusion protein can comprise a fragment of Zace1 that contains thecatalytic domain (e.g., a soluble Zace1 fragment) and an Fc fragment(e.g., a human Fc fragment).

In another variation, a Zace1 fusion protein comprises an IgG sequence,a Zace1 moiety covalently joined to the aminoterminal end of the IgGsequence, and a signal peptide that is covalently joined to theaminoterminal of the Zace1 moiety, wherein the IgG sequence consists ofthe following elements in the following order: a hinge region, a CH₂domain, and a CH₃ domain. Accordingly, the IgG sequence lacks a CH₁domain. The Zace1 moiety displays a Zace1 activity, as described herein,such as the ability to react with a substrate. This general approach toproducing fusion proteins that comprise both antibody and nonantibodyportions has been described by LaRochelle et al., EP 742830 (WO95/21258).

Fusion proteins comprising a Zace1 moiety and an Fc moiety can be used,for example, as an in vitro assay tool. For example, the presence of anZace1 substrate in a biological sample can be detected using aZace1-immunoglobulin fusion protein, in which the Zace1 moiety is usedto bind the substrate, and a macromolecule, such as Protein A or anti-Fcantibody, is used to bind the fusion protein to a solid support. Suchsystems can also be used to identify Zace1 substrates and inhibitors.

Other examples of antibody fusion proteins include polypeptides thatcomprise an antigen-binding domain and a Zace1 fragment that contains aZace1 catalytic domain. Such molecules can be used to target particulartissues for the benefit of Zace1 enzymatic activity.

The present invention further provides a variety of other polypeptidefusions. For example, a Zace1 polypeptide (corresponding to the C domainof somatic ACE) can be prepared as a fusion to an N domain of somaticACE. The native Zace1 signal sequence may also be recombinantlyexchanged with the signal sequence of somatic ACE or tACE. Likewise, thetransmembrane domain of Zace1 can be recombinantly exchanged with thatof somatic ACE or tACE. The catalytic domain of Zace1 can also berecombinantly exchanged for the corresponding region of somatic ACE,tACE, thermolysin or another zinc metalloprotease. Accordingly, part orall of a domain(s) conferring a biological function can be swappedbetween Zace1 of the present invention with the functionally equivalentdomain(s) from another family member, such as tACE or somatic ACE. Forexample, the region from His³⁹⁸ to Ser⁴³⁰ of Zace1 can be recombinantlyexchanged for the corresponding region of somatic ACE, tACE,thermolysin, or other zinc metalloprotease.

Polypeptide fusions can be expressed in recombinant host cells toproduce a variety of Zace1 fusion analogs. A Zace1 polypeptide can befused to two or more moieties or domains, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,for example, Tuan et al., Connective Tissue Research 34:1 (1996).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a domain(s) conferring a biologicalfunction can be swapped between Zace1 of the present invention with thefunctionally equivalent domain(s) from another family member, such astACE or somatic ACE. Such domains include, but are not limited to, thesecretory signal sequence, conserved motifs (such as the HEXXH andEX(I/V)X(D/S) domains), and the transmembrane or intracellular domain.Such fusion proteins would be expected to have a biological functionalprofile that is the same or similar to polypeptides of the presentinvention or other known zinc metalloprotease family proteins, dependingon the fusion constructed. General methods for enzymatic and chemicalcleavage of fusion proteins are described, for example, by Ausubel(1995) at pages 16-19 to 16-25.

The present invention also contemplates chemically modified Zace1compositions, in which a Zace1 polypeptide is linked with a polymer.Examples of suitable Zace1 polypeptides include soluble polypeptidesthat lack a functional transmembrane domain. Typically, the polymer iswater soluble so that the Zace1 conjugate does not precipitate in anaqueous environment, such as a physiological environment. An example ofa suitable polymer is one that has been modified to have a singlereactive group, such as an active ester for acylation, or an aldehydefor alkylation, In this way, the degree of polymerization can becontrolled. An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C1-C10) alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce Zace1 conjugates.

Zace1 conjugates used for therapy should preferably comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A Zace1conjugate can also comprise a mixture of such water-soluble polymers.

One example of a Zace1 conjugate comprises a Zace1 moiety and apolyalkyl oxide moiety attached to the N-terminus of the Zace1 moiety.PEG is one suitable polyalkyl oxide. As an illustration, Zace1 can bemodified with PEG, a process known as “PEGylation.” PEGylation of Zace1can be carried out by any of the PEGylation reactions known in the art(see, for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)). For example, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, Zace1 conjugates are formedby condensing activated PEG, in which a terminal hydroxy or amino groupof PEG has been replaced by an activated linker (see, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a Zace1 polypeptide. An example of an activatedPEG ester is PEG esterified to N-hydroxysuccinimide. As used herein, theterm “acylation” includes the following types of linkages between Zace1and a water soluble polymer: amide, carbamate, urethane, and the like.Methods for preparing PEGylated Zace1 by acylation will typicallycomprise the steps of (a) reacting a Zace1 polypeptide with PEG (such asa reactive ester of an aldehyde derivative of PEG) under conditionswhereby one or more PEG groups attach to Zace1, and (b) obtaining thereaction product(s). Generally, the optimal reaction conditions foracylation reactions will be determined based upon known parameters anddesired results. For example, the larger the ratio of PEG:Zace1, thegreater the percentage of polyPEGylated Zace1 product.

The product of PEGylation by acylation is typically a polyPEGylatedZace1 product, wherein the lysine ε-amino groups are PEGylated via anacyl linking group. An example of a connecting linkage is an amide.Typically, the resulting Zace1 will be at least 95% mono-, di-, ortri-pegylated, although some species with higher degrees of PEGylationmay be formed depending upon the reaction conditions. PEGylated speciescan be separated from unconjugated Zace1 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with Zace1 in the presence of a reducing agent. PEGgroups are preferably attached to the polypeptide via a —CH₂—NH group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of Zace1monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer Zace1 conjugate molecule can comprise the steps of: (a)reacting a Zace1 polypeptide with a reactive PEG under reductivealkylation conditions at a pH suitable to permit selective modificationof the α-amino group at the amino terminus of the Zace1, and (b)obtaining the reaction product(s). The reducing agent used for reductivealkylation should be stable in aqueous solution and preferably be ableto reduce only the Schiff base formed in the initial process ofreductive alkylation. Preferred reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer Zace1conjugates, the reductive alkylation reaction conditions are those whichpermit the selective attachment of the water soluble polymer moiety tothe N-terminus of Zace1. Such reaction conditions generally provide forpKa differences between the lysine amino groups and the α-amino group atthe N-terminus. The pH also affects the ratio of polymer to protein tobe used. In general, if the pH is lower, a larger excess of polymer toprotein will be desired because the less reactive the N-terminalα-group, the more polymer is needed to achieve optimal conditions. Ifthe pH is higher, the polymer:Zace1 need not be as large because morereactive groups are available. Typically, the pH will fall within therange of about 3 to about 9, or about 3 to about 6.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to Zace1 will generally bein the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to Zace1 will be 1:1 to 20:1 for polyPEGylation,and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)).

7. Isolation of Zace1 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. Particular purified polypeptides aresubstantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zace1 purified from natural sources, syntheticZace1 polypeptides, and recombinant Zace1 polypeptides and fusion Zace1polypeptides purified from recombinant host cells. In general, ammoniumsulfate precipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/orf carbohydratemoieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zace1 isolation and purification can be devisedby those of skill in the art. For example, anti-Zace1 antibodies,obtained as described below, can be used to isolate large quantities ofprotein by immunoaffinity purification.

Moreover, methods for binding enzymes, such as Zace1, to substratesbound to support media are well known in the art. For example, thepolypeptides of the present invention can be isolated by exploitation oftheir homology to somatic ACE and tACE. These enzymes can be purified byaffinity chromatography using the ACE inhibitor lisinopril[N-[(S)-1-carboxy-3-phenylpropyl]-Lys-Pro] as the ligand affixed to asolid support. Improved purification yields can be obtained using a 28Å, rather than a 14 Å, spacer between ligand and solid support.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

Zace1 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. Zace1 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

8. Zace1 Analogs and Zace1 Inhibitors

One general class of Zace1 analogs are variants having an amino acidsequence that is a mutation of the amino acid sequence disclosed herein.Another general class of Zace1 analogs is provided by anti-idiotypeantibodies, and fragments thereof, as described below. Moreover,recombinant antibodies comprising anti-idiotype variable domains can beused as analogs (see, for example, Monfardini et al., Proc. Assoc. Am.Physicians 108:420 (1996)). Since the variable domains of anti-idiotypeZace1 antibodies mimic Zace1, these domains can provide Zace1 enzymaticactivity. Methods of producing anti-idiotypic catalytic antibodies areknown to those of skill in the art (see, for example, Joron et al., Ann.N Y Acad. Sci. 672:216 (1992), Friboulet et al., Appl. Biochem.Biotechnol. 47:229 (1994), and Avalle et al., Ann. N Y Acad. Sci.864:118 (1998)).

Another approach to identifying Zace1 analogs is provided by the use ofcombinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al., Phage Display of Peptides and Proteins (Academic Press1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

One illustrative in vitro use of Zace1 and its analogs is the productionof labeled angiotensin II. For example, angiotensin I, radiolabeled atits N-terminus, can be incubated in the presence of Zace1 or an activevariant Zace1. The product of the reaction will be radiolabeledangiotensin II. This radiolabeled molecule can be used to study themetabolism of angiotensin II in vitro or to observe the tissuedistribution of administered angiotensin II in vivo.

The activity of Zace1 molecules of the present invention can be measuredusing a variety of assays that measure catalytic activity of the enzymein the presence or absence of zinc, or that measure the effects ofchloride or other monoanions on the catalytic activity of Zace1. Inaddition, the Zace1 polypeptides can be characterized by measuring thezinc content of these polypeptides. Radiolabeled ACE inhibitors areuseful for detecting high-affinity binding sites in zinc metalloproteasefamily members. One or more mutations of putative critical or importantresidues, in conjunction with known assays of ACE activity, permitanalysis of mutational effects on Zace1 structure, enzyme activity, andimmunological activity. In addition, both synthetic and natural ACEsubstrates can be useful in characterizing variant or mutated Zace1polypeptides. Studies that examine the interaction of Zace1 andcompetitive ACE inhibitors also can be employed to assay andcharacterize Zace1 polypeptides. Such assays are well known in the art.For a general reference, see Corvol et al., Meth. Enzymol. 246:283(1995). See also Williams et al., J. Biol. Chem. 269:29430 (1994),Sturrock et al., Biochem. 35:9560 (1996), and Michaud et al., Molec.Pharmacol. 51:1070 (1997).

As an illustration, a Zace1 variant can be tested for ACE activity usinghippuryl-L-histidyl-L-leucine (Hip-His-Leu) as a substrate (see, forexample, Sen et al., J. Biol. Chem. 268:25748 (1993)). In one version ofthis assay, a solubilized test polypeptide is incubated in 0.4 M sodiumborate buffer (pH 8.3) containing 300 mM sodium chloride for about 15 to30 minutes at 37° C. in the presence of varying concentrations ofHip-His-Leu (e.g., 0.4 to 5 mM). The amount of His-Leu liberated by thetest polypeptide is measured fluorometrically. Hip-His-Leu can also beused to identify Zace1 inhibitors by measuring the suppression of thecleavage of the substrate.

Other ACE substrates are known to those of skill in the art. Forexample, Isaac et al., Biochem. J. 328:587 (1997), have shown thatpolypeptides having Lys/Arg-Arg at the C-terminus are high-affinitysubstrates for human tACE. Another useful substrate to measure ACEactivity is [³H]benzol-Phe-Ala-Pro (Howell et al., Am. J. Physiol.258:L188 (1990)).

Solid phase systems can also be used to identify a substrate orinhibitor of a Zace1 polypeptide. For example, a Zace1 polypeptide,which may or may not be catalytically active, or Zace1 fusion proteincan be immobilized onto the surface of a receptor chip of a commerciallyavailable biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden).The use of this instrument is disclosed, for example, by Karlsson,Immunol. Methods 145:229 (1991), and Cunningham and Wells, J. Mol. Biol.234:554 (1993).

In brief, a Zace1 polypeptide or fusion protein is covalently attached,using amine or sulfhydryl chemistry, to dextran fibers that are attachedto gold film within a flow cell. A test sample is then passed throughthe cell. If a Zace1 substrate or inhibitor is present in the sample, itwill bind to the immobilized polypeptide or fusion protein, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination on- and off-rates, from which binding affinity can becalculated, and assessment of the stoichiometry of binding, as well asthe kinetic effects of Zace1 mutation. This system can also be used toexamine antibody-antigen interactions, and the interactions of othercomplement/anti-complement pairs.

Zace1 polypeptides can also be immobilized on a solid support, such asbeads of agarose, cross-linked agarose, glass, cellulosic resins,silica-based resins, polystyrene, cross-linked polyacrylamide, or likematerials that are stable under the conditions of use. Methods forlinking polypeptides to solid supports are known in the art, and includeamine chemistry, cyanogen bromide activation, N-hydroxysuccinimideactivation, epoxide activation, sulfhydryl activation, and hydrazideactivation. The resulting medium will generally be configured in theform of a column, and fluids containing substrate or putative substrateare passed through the column one or more times to allow substrate tobind to the Zace1 polypeptide. The substrate is then eluted usingchanges in salt concentration, chaotropic agents (guanidine HCl), or pHto disrupt substrate-Zace1 binding.

Accordingly, polypeptides of the present invention are useful as targetsfor identifying modulators of zinc protease activity. More particularly,Zace1 polypeptides are useful for screening or identifying new ACEinhibitors. The Zace1 polypeptides can also be used as a basis forrational drug design of inhibitory molecules. These newly identifiedinhibitory molecules may be more specific or more potent than known ACEinhibitors. Moreover, Zace1 inhibitors may exhibit a more favorable sideeffect profile than known ACE inhibitors. For example, Zace1 maycontribute to certain unwanted side effects of ACE inhibitors, and assuch, Zace1 would be useful to identify more specific ACE inhibitors.

In addition, inhibitory molecules identified using Zace1 polypeptides asa target may modulate different biological or physiological activitiesthan known ACE inhibitors (e.g., the inhibitors may be effective fordisorders other than those related to blood pressure and water and salthomeostasis). Zace1 inhibitors may provide broader inhibition than justACE inhibition (for instance, these inhibitors may modulate manymetalloprotease family members). Because Zace1 is more closelyhomologous to tACE than somatic ACE, Zace1 may permit selection ofdomain-specific inhibitors (those that inhibit the active sitecorresponding to the C domain of somatic ACE). Thus, a Zace1 inhibitormay specifically target angiotensin I and bradykinin-mediated effects,but have minimal or no effect on regulating hematopoiesis. Zace1inhibitors may beneficially improve the status of patients withcardiovascular disease, and atherosclerotic vascular disease inparticular, or renal disease, and diabetic nephropathy in particular.The effects of Zace1 inhibitors can be measured in vitro using culturedcells or in vivo by administering molecules of the claimed invention tothe appropriate animal model.

The measurement of Zace1 enzyme activity can also be used for diagnosis.For example, the measurement of serum ACE activity levels providesuseful information for the diagnosis of sarcoidosis and response totreatment (Studdy, Lancet 2(8104-5):1331 (1978)).

9. Production of Antibodies to Zace1 Proteins

Antibodies to Zace1 can be obtained, for example, using the product of aZace1 expression vector or Zace1 isolated from a natural source as anantigen. Particularly useful anti-Zace1 antibodies “bind specifically”with Zace1. Antibodies are considered to be specifically binding if theantibodies exhibit at least one of the following two properties: (1)antibodies bind to Zace1 with a threshold level of binding activity, and(2) antibodies do not significantly cross-react with polypeptidesrelated to Zace1.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zace1 polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)).

With regard to the second characteristic, antibodies do notsignificantly cross-react with related polypeptide molecules, forexample, if they detect Zace1, but not presently known polypeptidesusing a standard Western blot analysis. Examples of known relatedpolypeptides are known angiotensin converting enzymes, such as humansomatic ACE and tACE. A variety of assays known to those skilled in theart can be utilized to detect antibodies which specifically bind toZace1 proteins or peptides. Exemplary assays are described, for example,by Harlow and Lane (Eds.), Antibodies: A Laboratory Manual (Cold SpringHarbor Laboratory Press 1988). Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant Zace1 protein or polypeptide.

Anti-Zace1 antibodies can be produced using antigenic Zace1epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, preferably between 15 to about 30 amino acids containedwithin SEQ ID NO:1. However, peptides or polypeptides comprising alarger portion of an amino acid sequence of the invention, containingfrom 30 to 50 amino acids, or any length up to and including the entireamino acid sequence of a polypeptide of the invention, also are usefulfor inducing antibodies that bind with Zace1. It is desirable that theamino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues, while hydrophobic residues arepreferably avoided). Moreover, amino acid sequences containing prolineresidues may be also be desirable for antibody production.

As an illustration, potential antigenic sites in Zace1 were identifiedusing the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988),as implemented by the PROTEAN program (version 3.14) of LASERGENE(DNASTAR; Madison, Wis.). Default parameters were used in this analysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Garnier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:1 would provide suitable antigenic peptides:amino acids 28 to 34 (“antigenic peptide 1”), amino acids 39 to 56(“antigenic peptide 2”), amino acids 85 to 92 (“antigenic peptide 3”),amino acids 117 to 125 (“antigenic peptide 4”), amino acids 132 to 147(“antigenic peptide 5”), amino acids 233 to 245 (“antigenic peptide 6”),amino acids 376 to 394 (“antigenic peptide 7”), amino acids 512 to 523(“antigenic peptide 8”), amino acids 580 to 586 (“antigenic peptide 9”),amino acids 635 to 649 (“antigenic peptide 10”), and amino acids 655 to662 (“antigenic peptide 11”). The present invention contemplates the useof any one of antigenic peptides 1 to 11 to generate antibodies toZace1. The present invention also contemplates polypeptides comprisingat least one of antigenic peptides 1 to 11.

Polyclonal antibodies to recombinant Zace1 protein or to Zace1 isolatedfrom natural sources can be prepared using methods well-known to thoseof skill in the art. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press 1992), and Williams et al., “Expression of foreignproteins in E. coli using plasmid vectors and purification of specificpolyclonal antibodies,” in DNA Cloning 2: Expression Systems, 2ndEdition, Glover et al. (eds.), page 15 (Oxford University Press 1995).The immunogenicity of a Zace1 polypeptide can be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of Zace1 or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like,” suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-Zace1 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zace1 antibodies can be generated. Rodentmonoclonal antibodies to specific antigens may be obtained by methodsknown to those skilled in the art (see, for example, Kohler et al.,Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zace1 gene product, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zace1 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal, “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zace1 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZace1 polypeptide in vitro, and selecting antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled Zace1 protein or peptide). Genes encoding polypeptides havingpotential Zace1 polypeptide binding domains can be obtained by screeningrandom peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zace1 sequences disclosed herein to identifyproteins which bind to Zace1.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zace1 antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain. Typical residues of human antibodies are then substituted in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by Orlandi et al., Proc. Nat'l Acad.Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522(1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992),Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J. Immun.150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (HumanaPress, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zace1 antibodies or antibody fragments, using standardtechniques. See, for example, Green et al., “Production of PolyclonalAntisera,” in Methods In Molecular Biology: Immunochemical Protocols,Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can beprepared using anti-Zace1 antibodies or antibody fragments as immunogenswith the techniques, described above. As another alternative, humanizedanti-idiotype antibodies or subhuman primate anti-idiotype antibodiescan be prepared using the above-described techniques. Methods forproducing anti-idiotype antibodies are described, for example, by Irie,U.S. Pat. No. 5,208,146, Greene, et. al., U.S. Pat. No. 5,637,677, andVarthakavi and Minocha, J. Gen. Virol. 77:1875 (1996).

10. Use of Zace1 Nucleotide Sequences to Detect Gene Expression and GeneStructure

Nucleic acid molecules can be used to detect the expression of a Zace1gene in a biological sample. Probe molecules may be DNA, RNA,oligonucleotides, and the like. As used herein, the term “portion”refers to at least eight nucleotides to at least 20 or more nucleotides.Preferred probes bind with regions of the Zace1 gene that have a lowsequence similarity to comparable regions in other proteins, such asother angiotensin converting enzymes.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget Zace1 RNA species. After separating unbound probe from hybridizedmolecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, Zace1 RNAcan be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zace1 oligonucleotide probes are also useful for in vivo diagnosis. Asan illustration, ¹⁸F-labeled oligonucleotides can be administered to asubject and visualized by positron emission tomography (Tavitian et al.,Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

Preferably, PCR primers are designed to amplify a portion of the Zace1gene that has a low sequence similarity to a comparable region in otherproteins, such as other angiotensin converting enzymes.

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withZace1 primers (see, for example, Wu et al. (eds.), “Rapid Isolation ofSpecific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages15-28 (CRC Press, Inc. 1997)). PCR is then performed and the productsare analyzed using standard techniques.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zace1 probe, and examinedby autoradiography. Additional alternative approaches include the use ofdigoxigenin-labeled deoxyribonucleic acid triphosphates to providechemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach for detection of Zace1 expression is cycling probetechnology (CPT), in which a single-stranded DNA target binds with anexcess of DNA-RNA-DNA chimeric probe to form a complex, the RNA portionis cleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zace1 sequences can utilize approaches such asnucleic acid sequence-based amplification (NASBA), cooperativeamplification of templates by cross-hybridization (CATCH), and theligase chain reaction (LCR) (see, for example, Marshall et al., U.S.Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161 (1996),Ehricht et al., Eur. J. Biochem. 243:358 (1997), and Chadwick et al., J.Virol. Methods 70:59 (1998)). Other standard methods are known to thoseof skill in the art.

Zace1 probes and primers can also be used to detect and to localizeZace1 gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)). Suitable test samples include blood, urine,saliva, tissue biopsy, and autopsy material.

Clinically significant polymorphisms of the human ACE gene have beendiscovered (see, for example, Matsusaka and Ichikawa, Annu. Rev.Physiol. 59:395 (1997)). A polymorphism associated with intron 16 isassociated with plasma and intracellular levels of ACE, as well asincreased risk of myocardial infarction. ACE polymorphisms are alsoassociated with progression to chronic renal failure in IgA nephropathy,and diabetic nephropathy (Marre et al., Diabetes 43:384 (1994); Yoshidaet al., J. Clin. Invest. 96:2162 (1995)). Other ACE gene mutations areassociated with the risk of developing cardiovascular disease (Raynoldsand Perryman, U.S. Pat. No. 5,800, 990).

Nucleic acid molecules comprising Zace1 nucleotide sequences can be usedto determine whether a subject's chromosomes contain a mutation in theZace1 gene. Detectable chromosomal aberrations at the Zace1 gene locusinclude, but are not limited to, aneuploidy, gene copy number changes,insertions, deletions, restriction site changes and rearrangements. Ofparticular interest are genetic alterations that inactivate the Zace1gene.

Aberrations associated with the Zace1 locus can be detected usingnucleic acid molecules of the present invention by employing moleculargenetic techniques, such as restriction fragment length polymorphism(RFLP) analysis, short tandem repeat (STR) analysis employing PCRtechniques, amplification-refractory mutation system analysis (ARMS),single-strand conformation polymorphism (SSCP) detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis (FAMA), and other genetic analysis techniques known inthe art (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)).

The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al, Blood 91:3920 (1998)). According to this approach,RNA is isolated from a biological sample, and used to synthesize cDNA.PCR is then used to amplify the Zace1 target sequence and to introducean RNA polymerase promoter, a translation initiation sequence, and anin-frame ATG triplet. PCR products are transcribed using an RNApolymerase, and the transcripts are translated in vitro with aT7-coupled reticulocyte lysate system. The translation products are thenfractionated by SDS-PAGE to determine the lengths of the translationproducts. The protein truncation test is described, for example, byDracopoli et al. (eds.), Current Protocols in Human Genetics, pages9.11.1-9.11.18 (John Wiley & Sons 1998). Protein truncation can also beexamined by comparing Zac1 protein isolated from a subject with apolypeptide comprising the amino acid sequence disclosed herein.

Localization of the chromosomal location of the Zace1 gene can beachieved using radiation hybrid mapping, which is a somatic cell genetictechnique developed for constructing high-resolution, contiguous maps ofmammalian chromosomes (Cox et al., Science 250:245 (1990)). Partial orfull knowledge of a gene's sequence allows one to design PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Radiation hybrid mapping panels are commercially available which coverthe entire human genome, such as the Stanford G3 RH Panel and theGeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, Ala.). Thesepanels enable rapid, PCR-based chromosomal localizations and ordering ofgenes, sequence-tagged sites, and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers.

The present invention also contemplates kits for performing a diagnosticassay for Zace1 gene expression or to detect-mutations in the Zace1gene. Such kits comprise nucleic acid probes, such as double-strandednucleic acid molecules, as well as single-stranded nucleic acidmolecules. Probe molecules may be DNA, RNA, oligonucleotides, and thelike. Kits may comprise nucleic acid primers for performing PCR. A kitwill comprise at least one container comprising a Zace1 probe or primer.The kit may also comprise a second container comprising one or morereagents capable of indicating the presence of Zace1 sequences. Examplesof such indicator reagents include detectable labels such as radioactivelabels, fluorochromes, chemiluminescent agents, and the like. A kit mayalso comprise a means for conveying to the user that the Zace1 probesand primers are used to detect Zace1 gene expression. For example,written instructions may state that the enclosed nucleic acid moleculescan be used to detect either a nucleic acid molecule that encodes Zace1,or a nucleic acid molecule having a nucleotide sequence that iscomplementary to a Zace1-encoding nucleotide sequence. The writtenmaterial can be applied directly to a container, or the written materialcan be provided in the form of a packaging insert.

11. Use of Anti-Zace1 Antibodies to Detect Zace1

Antibodies to Zace1 can be used for tagging cells that express Zace1,for isolating Zace1 or portions thereof by affinity purification, fordiagnostic assays for determining circulating levels of Zace1polypeptides, for detecting or quantitating Zace1 as a marker ofunderlying pathology or disease, in analytical methods employing FACS,for screening expression libraries, for generating anti-idiotypicantibodies, and as neutralizing antibodies or as antagonists to blockZace1 effects in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to Zace1 orfragments thereof can be used in vitro to detect denatured Zace1 orfragments thereof in assays, for example, Western Blots or other assaysknown in the art.

Accordingly, the present invention contemplates the use of anti-Zace1antibodies to screen biological samples in vitro for the presence ofZace1. In one type of in vitro assay, anti-Zace1 antibodies are used inliquid phase. For example, the presence of Zace1 in a biological samplecan be tested by mixing the biological sample with a trace amount oflabeled Zace1 and an anti-Zace1 antibody under conditions that promotebinding between Zace1 and its antibody. Complexes of Zace1 andanti-Zace1 in the sample can be separated from the reaction mixture bycontacting the complex with an immobilized protein which binds with theantibody, such as an Fc antibody or Staphylococcus protein A. Theconcentration of Zace1 in the biological sample will be inverselyproportional to the amount of labeled Zace1 bound to the antibody anddirectly related to the amount of free labeled Zace1. Illustrativebiological samples include blood, urine, saliva, tissue biopsy, andautopsy material.

Alternatively, in vitro assays can be performed in which anti-Zace1antibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zace1 antibodies can be used to detect Zace1in tissue sections prepared from a biopsy specimen. Such immunochemicaldetection can be used to determine the relative abundance of Zace1 andto determine the distribution of Zace1 in the examined tissue. Generalimmunochemistry techniques are well established (see, for example,Ponder, “Cell Marking Techniques and Their Application,” in MammalianDevelopment: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods InMolecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press,Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zace1 antibody, and then contacting the biologicalsample with a detectably labeled molecule which binds to the antibody.For example, the detectably labeled molecule can comprise an antibodymoiety that binds to anti-Zace1 antibody. Alternatively, the anti-Zace1antibody can be conjugated with avidin/streptavidin (or biotin) and thedetectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an anti-Zace1 antibody can be conjugated with adetectable label to form an anti-Zace1 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-Zace1 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zace1 immunoconjugates can be detectably labeled bycoupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zace1immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zace1 immunoconjugates can be detectably labeled bylinking an anti-Zace1 antibody component to an enzyme. When theanti-Zace1-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zace1 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs etal., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l J. Cancer 46:1101(1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zace1 antibodies that have been conjugatedwith avidin, streptavidin, and biotin (see, for example, Wilchek et al.(eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184(Academic Press 1990), and Bayer et al., “Immunochemical Applications ofAvidin-Biotin Technology,” in Methods In Molecular Biology, Vol. 10,Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).

Methods for performing inmunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

The present invention also contemplates kits for performing animmunological diagnostic assay for Zace1 gene expression. Such kitscomprise at least one container comprising an anti-Zace1 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zace1antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zace1 antibodies or antibody fragments areused to detect Zace1 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect Zace1. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

12. Bioactive Conjugates of Zace1 Polypeptides and Antibodies

The present invention includes antibodies or polypeptides that aredirectly or indirectly conjugated to drugs, toxins, radionuclides andthe like, which can be used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (substrate,receptor, or antigen, respectively, for instance). More specifically,Zace1 polypeptides or anti-Zace1 antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that expressthe anti-complementary molecule.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Polypeptides orantibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

In another aspect of the invention, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a substrate and/or ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the domain only fusion protein includes acomplementary molecule, the anti-complementary molecule can beconjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, Zace1-cytokine fusion proteins oranti-Zace1-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues, if the Zace1 polypeptide or anti-Zace1antibody targets the hyperproliferative target cell. For example,Hornick et al., Blood 89:4437 (1997), described fusion proteins thatenable targeting of a cytokine to a desired site of action, therebyproviding an elevated local concentration of cytokine. Suitablecytokines for this purpose include interleukin-2 andgranulocyte-macrophage colony-stimulating factor (GM-CSF), and otherimmunomodulators, for instance. Suitable Zace1 polypeptides oranti-Zace1 antibodies target an undesirable cell or tissue (i.e., ahyperproliferative vascular epithelial cell or a transformed cell). Suchpolypeptide or antibody can be conjugated with a radionuclide, andparticularly with a β-emitting radionuclide, to reduce restenosis ortransformed cell mass. Such therapeutic approach poses less danger toclinicians who administer the radioactive therapy. For instance,iridium-192 impregnated ribbons placed into stented vessels of patientsuntil the required radiation dose was delivered showed decreased tissuegrowth in the vessel and greater luminal diameter than the controlgroup, which received placebo ribbons. Further, revascularisation andstent thrombosis were significantly lower in the treatment group.Similar results are predicted with targeting of a bioactive conjugatecontaining a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intraarterially or intraductally, or may beintroduced locally at the intended site of action. Suitable modes ofadministration of therapeutic proteins are described below.

13. Therapeutic Uses of Polypeptides Having Zace1 Activity

The present invention includes the use of proteins, polypeptides, andpeptides having Zace1 activity (such as Zace1 polypeptides (e.g.,soluble forms of Zace1), Zace1 analogs (e.g., anti-Zace1 anti-idiotypeantibodies), and Zace1 fusion proteins) to a subject who lacks anadequate amount of this polypeptide. In contrast, Zace1 antagonists(e.g., anti-Zace1 antibodies) can be used to treat a subject whoproduces an excess of Zace1.

The kallikrein-kinin (contact) system modulates therenin-angiotensin-aldosterone system, prostaglandins, vasopressins,sodium-water balance, renal hemodynamics, and blood pressure. Stadnickiet al., FASEB J. 12:325 (1998), have shown that a reversible inhibitorof plasma kallikrein decreased chronic intestinal inflammation in anexperimental model relevant to Crohn's disease. One of the actions ofkallikrein is to cleave high molecular weight kininogen to producebradykinin, a peptide that enhances vasodilation, increases vascularpermeability, and influences intestinal motility and electrolytesecretion (see, for example, Bhoola et al., Pharmacol. Rev. 44:1(1992)). The inhibition of kallikrein by the reversible inhibitor,therefore, should decrease bradykinin activity levels, which isconsistent with evidence that kinins mediate gastrointestinalinflammation associated with inflammatory bowel disease, such as Crohn'sdisease (see, for example, Bachvarov et al., Gastroenterology 115:1045(1998)).

ACE also decreases bradykinin activity by cleaving the peptide.Accordingly, decreased ACE activity should be correlated with increasedbradykinin activity. Studies have shown that serum ACE activity issignificantly lowered in certain patients who have active Crohn'sdisease (see, for example, Silverstein et al., Am. J. Clin. Pathol.75:175 (1981); Sommer et al., Enzyme 35:181 (1986)). Taken together,these observations indicate that ACE can be used to treat conditionsassociated with inflammation, such as inflammatory bowel disease.

The present invention therefore includes the use of polypeptides havingZace1 activity (e.g., Zace1 polypeptides, functional fragments of Zace1,anti-Zace1 anti-idiotype antibodies, etc.) to treat an inflammatorybowel disease (e.g., Crohn's disease and ulcerative colitis). Moregenerally, the present invention includes the use of polypeptides havingZace1 activity to treat diseases associated with inflammation, such asarthritis and enterocolitis, two conditions which have been treated witha kallikrein inhibitor (see, for example, DeLa Cadena et al., FASEB J.9:446 (1995); Stadnicki et al., Dig. Dis. Sci. 41:912 (1996)). Methodsfor identification of subjects suitable for such treatment are wellknown to those of skill in the art (see, for example, Rakel (ed.),Conn's 1999 Current Therapy (W.B. Saunders Company 1999)).

Generally, the dosage of administered Zace1 (or Zace1 analog or fusionprotein) will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of Zace1 which is in the range of from about 1 pg/kg to 10 mg/kg(amount of agent/body weight of patient), although a lower or higherdosage also may be administered as circumstances dictate.

Administration of a molecule having Zace1 activity to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. Regional administrationis particularly useful for treatment of an inflammatory bowel disease.When administering therapeutic proteins by injection, the administrationmay be by continuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising Zace1 canbe prepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). Thisapproach is illustrated by the AERX diabetes management system, which isa hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer a molecule having Zace1 activity (Potts et al.,Pharm. Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zace1 activity can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic proteins are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers, such as 5%dextrose in water, are well-known to those in the art. Formulations canfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, by Gennaro (ed.), Remington's Pharmaceutical Sciences, 19thEdition (Mack Publishing Company 1995).

For purposes of therapy, molecules having Zace1 activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zace1 activity and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient patient.For example, common symptoms of Crohn's disease include chronic diarrheawith abdominal pain, fever, anorexia, weight loss, and a right lowerquadrant mass. An agent used to treat Crohn's disease is physiologicallysignificant if its presence alleviates at least one of these symptoms.

A pharmaceutical composition comprising Zace1 (or Zace1 analog or fusionprotein) can be furnished in liquid form, in an aerosol, or in solidform. Liquid forms, are illustrated by injectable solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides having Zace1 activity can be encapsulated within liposomesusing standard techniques of protein microencapsulation (see, forexample, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al, Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly(ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving Zace1 activity and Zace1 antagonists, in which a polypeptide islinked with a polymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a molecule having Zace1 activityor a Zace1 antagonist (e.g., an antibody or antibody fragment that bindsa Zace1 polypeptide). Therapeutic polypeptides can be provided in theform of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticpolypeptide. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition. Moreover, suchinformation may include a statement that the Zace1 composition iscontraindicated in patients with known hypersensitivity to Zace1.

14. Therapeutic Uses of Zace1 Nucleotide Sequences

The present invention includes the use of Zace1 nucleotide sequences toprovide Zace1 to a subject in need of such treatment. In addition, atherapeutic expression vector can be provided that inhibits Zace1 geneexpression, such as an anti-sense molecule, a ribozyme, or an externalguide sequence molecule.

There are numerous approaches to introduce a Zace1 gene to a subject,including the use of recombinant host cells that express Zace1, deliveryof naked nucleic acid encoding Zace1, use of a cationic lipid carrierwith a nucleic acid molecule that encodes Zace1, and the use of virusesthat express Zace1, such as recombinant retroviruses, recombinantadeno-associated viruses, recombinant adenoviruses, and recombinantHerpes simplex viruses (see, for example, Mulligan, Science 260:926(1993), Rosenberg et al., Science 242:1575 (1988), LaSalle et al.,Science 259:988 (1993), Wolff et al., Science 247:1465 (1990),Breakfield and Deluca, The New Biologist 3:203 (1991)). In an ex vivoapproach, for example, cells are isolated from a subject, transfectedwith a vector that expresses a Zace1 gene, and then transplanted intothe subject.

In order to effect expression of a Zace1 gene, an expression vector isconstructed in which a nucleotide sequence encoding a Zace1 gene isoperably linked to a core promoter, and optionally a regulatory element,to control gene transcription. The general requirements of an expressionvector are described above.

Alternatively, a Zace1 gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N.Y. Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant herpes simplex virus can beprepared in Vero cells, as described by Brandt et al., J. Gen. Virol.72:2043 (1991), Herold et al., J. Gen. Virol. 75:1211 (1994), Visalliand Brandt, Virology 185:419 (1991), Grau et al., Invest. Ophthalmol.Vis. Sci. 30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992),and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press1997).

Alternatively, an expression vector comprising a Zace1 gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration. Forexample, Aihara and Miyazaki, Nature Biotechnology 16:867 (1998), havedemonstrated the use of in vivo electroporation for gene transfer intomuscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zace1 anti-sense RNA that inhibits the expression of Zace1.Suitable sequences for anti-sense molecules can be derived from thenucleotide sequences of Zace1 disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zace1 mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode aZace1 gene. According to this approach, an external guide sequence canbe constructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). Preferably, the external guide sequencecomprises a ten to fifteen nucleotide sequence complementary to Zace1mRNA, and a 3′-NCCA nucleotide sequence, wherein N is preferably apurine. The external guide sequence transcripts bind to the targetedmRNA species by the formation of base pairs between the mRNA and thecomplementary external guide sequences, thus promoting cleavage of mRNAby RNase P at the nucleotide located at the 5′-side of the base-pairedregion.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zace1 nucleotide acid sequence, such as a recombinant virus,will vary depending upon such factors as the subject's age, weight,height, sex, general medical condition and previous medical history.Suitable routes of administration of therapeutic vectors includeintravenous injection, intraarterial injection, intraperitonealinjection, intramuscular injection, intratumoral injection, andinjection into a cavity that contains a tumor. As an illustration,Horton et al., Proc. Nat'l Acad. Sci. USA 96:1553 (1999), demonstratedthat intramuscular injection of plasmid DNA encoding interferon-αproduces potent antitumor effects on primary and metastatic tumors in amurine model.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject. For example, common symptoms of Crohn's diseaseinclude chronic diarrhea with abdominal pain, fever, anorexia, weightloss, and a right lower quadrant mass. An agent used to treat Crohn'sdisease is physiologically significant if its presence alleviates atleast one of these symptoms.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

15. Production of Transgenic Mice

Transgenic mice can be engineered to over-express the Zace1 gene in alltissues or under the control of a tissue-specific or tissue-preferredregulatory element. These over-producers of Zace1 can be used tocharacterize the phenotype that results from over-expression, and thetransgenic animals can serve as models for human disease caused byexcess Zace1. Transgenic mice that over-express Zace1 also provide modelbioreactors for production of Zace1 in the milk or blood of largeranimals. Methods for producing transgenic mice are well-known to thoseof skill in the art (see, for example, Jacob, “Expression and Knockoutof Interferons in Transgenic Mice,” in Overexpression and Knockout ofCytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (AcademicPress, Ltd. 1994), Monastersky and Robl (eds.), Strategies in TransgenicAnimal Science (ASM Press 1995), and Abbud and Nilson, “RecombinantProtein Expression in Transgenic Mice,” in Gene Expression Systems:Using Nature for the Art of Expression, Fernandez and Hoeffler (eds.),pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZace1 gene can begin with adult, fertile males (studs) (B6C3f1, 2-8months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a Zace1 encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zace1encoding sequences can encode a polypeptide comprising the amino acidresidues of SEQ ID NO:1, or a fragment thereof.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductive,organs allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zace1 gene or aselectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zace1 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

In addition to producing transgenic mice that over-express Zace1, it isuseful to engineer transgenic mice with either abnormally low or noexpression of the gene. Such transgenic mice provide useful models fordiseases associated with a lack of Zace1. As discussed above, Zace1 geneexpression can be inhibited using anti-sense genes, ribozyme genes, orexternal guide sequence genes. To produce transgenic mice thatunder-express the Zace1 gene, such inhibitory sequences are targeted toZace1 mRNA. Methods for producing transgenic mice that have abnormallylow expression of a particular gene are known to those in the art (see,for example, Wu et al., “Gene Underexpression in Cultured Cells andAnimals by Antisense DNA and RNA Strategies,” in Methods in GeneBiotechnology, pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zace1 gene expression is to generate mice having at least one normalZace1 allele replaced by a nonfunctional Zace1 gene. One method ofdesigning a nonfunctional Zace1 gene is to insert another gene, such asa selectable marker gene, within a nucleic acid molecule that encodesZace1. Standard methods for producing these so-called “knockout mice”are known to those skilled in the art (see, for example, Jacob,“Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., “NewStrategies for Gene Knockout,” in Methods in Gene Biotechnology, pages339-365 (CRC Press 1997)).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An antibody that binds with a polypeptide having an amino acidsequence that consists of the amino acid sequence of SEQ ID NO:1.
 2. Theantibody of claim 1, wherein the antibody binds with amino acid residues52 to 662 of SEQ ID NO:1.
 3. The antibody of claim 1, wherein theantibody is a polyclonal antibody.
 4. The antibody of claim 1, whereinthe antibody is a monoclonal antibody.
 5. The antibody of claim 1,wherein the antibody is a chimeric antibody.
 6. The antibody of claim 1,wherein the antibody is a single chain antibody.
 7. The antibody ofclaim 1, wherein the antibody is a humanized antibody.
 8. An antibodyfragment that binds with a polypeptide having an amino acid sequencethat consists of the amino acid sequence of SEQ ID NO:1.
 9. The antibodyfragment of claim 8, wherein the antibody fragment binds with amino acidresidues 52 to 662 of SEQ ID NO:1.
 10. The antibody fragment of claim 8,wherein the antibody fragment is an F(ab)′₂ fragment.
 11. The antibodyfragment of claim 8, wherein the antibody fragment is a Fab fragment.12. The antibody fragment of claim 8, wherein the antibody fragment is aFv fragment.